Our long term objectives are to understand molecular determinants of virulence in pathogenic bacteria, using both structural and genetic approaches to explore functions and regulation of representative bacterial toxins. We will study diphtheria toxin (DT) and heat-labile enterotoxins (LTs), which are major virulence factors of Corynebacterium diphtheriae and enterotoxigenic Escherichia coli (ETEC), respectively. Synthesis of DT is controlled by the diphtheria toxin repressor (DtxR), the prototype for a new class of bacterial global regulatory proteins that includes a recently discovered functional homolog of DtxR in mycobacteria. Information from such studies provides a basis for rational design of improved vaccines, immunotoxins, hybrid toxins, related biologics, and chemotherapeutic agents. Our specific aims for the next grant cycle are to: 1) Characterize the diphtheria toxin repressor. We will determine the crystal structure of DtxR by X-ray diffraction, characterize its functional domains, construct a defined DtxR-negative mutant of C. diphtheriae, and screen for DNA sequences homologous with dtxR in coryneform bacteria and other related bacteria. 2) Characterize DtxR- regulated promoter/ operator regions and genes of C. diphtheriae. We will clone a set of DtxR-regulated promoter/operators from C. diphtheriae, identify conserved features, and use site-directed mutagenesis to probe operator function. We will clone and characterize genes of C. diphtheriae that are regulated by these promoter/operators to identify the proteins, pathways, and functions included in the DtxR regulon. 3) Characterize the mycobacterial homolog of DtxR. We will purify the functional DtxR homolog from M. tuberculosis, determine its metal ion-binding and DNA-binding specificities, crystallize it, and determine its structure. We will use molecular modeling to design potential new chemotherapeutic agents that will enhance activity of the DtxR homolog and inhibit growth of M. tuberculosis and other pathogenic mycobacteria under low-iron conditions in vivo. 4) Characterize type II heat-labile enterotoxins of E. coli. We will determine crystal structures of LT-IIa and LT-IIb, compare their toxicities in several animal models, and test their immunogenicity and adjuvanticity in mice. We will develop improved vectors for construction of holotoxin-like chimeras and for efficient secretion of foreign proteins to the periplasm in E. coli and explore the use of these vectors and type II enterotoxins for development of new oral vaccines against pathogenic microbes.